Akt
Akt (Protein kinase B) is a serine/threonine protein kinase that is known to be involved in diverse cellular processes including proliferation, motility, growth, glucose homeostasis, survival and cell death. Akt is one of the three principal components of the PI3K/Akt pathway (phosphatdylinositol 3-kinase, its antagonist PTEN and Akt). Mutation in components of this pathway are among the most frequently observed mutations in cancers and are found in up to 70% of breast cancers. In humans, there are three Akt family members, Akt 1, Akt 2 and Akt3 which are transcribed from different genes. The majority of research publications on Akt refer either to Akt1 or to Akt without specifying which family member, a consequence of the widespread use of pan-Akt antibodies that do not distinguish between the family members. Of the three isoforms, least is known about Akt3. Indeed, in a recent review article “Key signalling nodes in mammary gland development and cancer. Signalling downstream of PI3 kinase in mammary epithelium: a play in 3 Akts” (Wickenden J A and Watson C J, Breast Cancer Research 2010, 12, 202), Akt3 is mentioned just three times: once to establish its existence, once to note that it appears to have a minor role in normal mammary gland development and once to note that it does not affect Stat5a phosphorylation during pregnancy and lactation.
The roles for Akt1, Akt2 and Akt3 in normal development have been studied in knock-out mice, revealing that Akt1 is important for overall growth (knock-out mice are generally healthy but have reduced growth). Akt2 is primarily involved in glucose metabolism (knockout mice grow normally but show insulin resistance) and Akt3 is important in brain development (see e.g. Dummler B, Hemmings B A. Physiological roles of PKB/Akt isoforms in development and disease. Biochem Soc Trans 2007; 35:231-5). A more general role for Akt1 and Akt2 is suggested by their widespread expression throughout the body, while Akt3 has more restricted expression in the brain, kidney and heart.
Akt is considered an attractive target for cancer therapy, and inhibition of Akt alone or in combination with standard cancer chemotherapeutics has been postulated to reduce the apoptotic threshold and preferentially kill cancer cells (Lindley C W, Curr Top Med Chem, 10, 458, 2010). A recent review of attempts to inhibit Akt members pinpoints Akt2 as the most commonly mutated family member in cancers and suggests that inhibition of Akt1 and Akt2 would be optimal (Mattmann M E at al “Inhibition of Akt with small molecules and biologics: historical perspective and current status of the patent landscape”, Expert Opinion on Therapeutic Patents, 21, 1309, 2011). Many of the compounds covered in this review have poor selectivity for Akt compared to other kinases and generally focus on Akt1. Compounds reported in this review with selectivity between the different family members overwhelmingly inhibit Akt1 and/or Akt2 rather than Akt3.
Despite the overwhelming focus on Akt1 in the literature, Akt3 overexpression has been linked to several cancers including melanoma (Cancer Res. 2004 Oct. 1; 64(19):7002-10) and ovarian cancer (Cancer Discov. 2012 Jan. 1; 2(1):56-67).
Several patent publications relate to the use of Akt3.
WO2010/091354 (H Lee Moffat Cancer Institute, Inc.) relates to methods of diagnosing cancer in a subject involving determining levels of expression of Tyrosine 176-phosphorylated AKT1 rather than AKT3.
US20120040842 (Baker, at al.) lists Akt3 amongst a vast array of genes that may be assessed to determine the prognosis of colorectal cancer. However, Akt3 is not selected as a preferred marker.
US20120028264 (Shag, at al.) lists Akt3 {Table 3A} amongst a vast any of genes, expression levels of which may be determined in the assessing the likelihood of prostate cancer recurring in a subject. The significance of Akt3 is not specifically mentioned.
US20120021983 (Tsichlis, et al.) relates to a method of diagnosing or prognosing a potential cancer and progression of an existing cancer by assessing a subject's Akt isoform profile, especially the ratio of Akt1 to Akt2, by comparing that profile with a normal Akt isoform profile.
US20120003209 (The Translational Genomics Research Institute) relates to methods and kits used in the identification of invasive glioblastoma based upon the expression levels of Akt1 and Akt2. Akt3 mRNA expression was found to be high in non-neoplastic brain speciments and decreased in glial tumours. Furthermore Akt3 expression was found to be significantly higher in long term surviving patients.
U.S. Pat. No. 8,133,684 (Aebersold et al.) discloses methods of determining androgen responses in prostate cells, mentioning Akt3 in a long list of possible prostate cancer biomarkers.
The Epithelial-Mesenchymal Transition (EMT)
Epithelial tissues make up one of the four basic tissue types of the body, along with connective tissue, muscle and nervous tissue. Epithelial cells are characterised by a tendency to form into sheets of polarised cells held together by strong intercellular junctions. As a consequence of this, epithelial cells are not able to move freely and show little migration compared to other cell types. In contrast, mesenchymal-like cells (e.g. fibroblasts) lack strong intercellular junctions and can move as individual cells. They can be highly motile and able to migrate through the extracellular matrix.
The Epithelial-Mesenchymal Transition (EMT) is a natural cellular program in which individual epithelial cells lose the gene expression patterns and behaviours characteristic of epithelial cells and instead begin to look, behave and express genes typical of mesenchymal cells. In so doing they lose adhesion and apical-basal polarity and gain the ability to migrate and invade the extracellular matrix. EMT is not irreversible. A mirror process called Mesenchymal-Epithelial Transition (MET) results in the loss of mesenchymal characteristics and re-establishment of cell-cell adhesion and apical-basal polarity.
EMT is especially important during embryonic development. It plays a fundamental role in gastrulation, where an embryo consisting of a single epithelial cell layer develops into one with the three classical germ layers, ectoderm, mesoderm and endoderm. Slightly later in vertebrate development, EMT gives rise to the neural crest cells. These cells migrate throughout the embryo and give rise to many different structures including ganglia of the peripheral nervous system, bone and cartilage of the face and head, pigment cells and glial cells. Further rounds of MET and EMT are essential for the formation of internal organs from both the mesoderm and endoderm.
EMT and Disease
In contrast to its importance during embryonic development, the EMT program is seldom activated in healthy adults. It is, however, induced in response to inflammation following injury or disease: EMT plays a role in wound healing and tissue repair, and occurs during organ degenerative disease (e.g. renal fibrosis).
EMT is also increasingly understood to play a key role in cancer metastasis. Carcinomas are epithelial cancers, and, in order for metastasis to occur, individual cells must escape the primary tumour and undergo a series of migrations. These include migration from the primary tumour into the local circulatory or lymphatic system, and extravasation from the vasculature and establishment at the site of metastasis. There is now good and growing evidence that interactions between tumour cells and their microenvironment can lead to induction of EMT in some of the tumour cells. The resulting increased cell migration and invasion potential of these cells then enhances the likelihood of a metastasis becoming, established. The receptor tyrosin kinase Axl, which is a chronic myelogenous leukemia-associated oncogene, has recently been shown to be an essential EMT-induced effector in the invasion metastasis cascade (WO2010/103388).
As well as this role in increasing metastatic potential, the EMT program has recently been linked with Cancer Stein Cells (CSCs). These cells have been postulated to represent a subset of tumour cells with stem cell characteristics—i.e. the ability to give rise to all the cell types found in a particular cancer, and thus the ability to form a new tumour. Although they may represent only a tiny fraction of the cells in a tumour, CSCs are thought to be particularly resistant to existing anti-cancer drugs. Even though drug treatment may kill the vast majority of cells in the tumour, a single surviving CSC can therefore lead to a relapse of the disease. Recent evidence suggests an overlap between EMT and CSC phenotypes, suggesting that EMT may also play a role in recurrence of cancer after chemotherapy and the development of drug-resistant tumours.
Robust biomarkers for the EMT phenotype would be useful in identifying patients at particular risk of developing metastatic or drug-resistant cancer, while novel drugs that target cells that have undergone EMT will reduce metastasis and relapse following conventional therapy.
EMT activators (e.g. the transcription factor Slug) increase Akt1 activity/expression. It is also known that Akt1 activation (for example of the myristylated variant MyrAkt1) induces EMT activators (e.g. the transcriptional repressor, Snail; Oncogene. 2007 Nov. 22; 26(53):7445-56. Epub 2007 Jun. 1) and also causes biomarker switching from epithelial to mesenchymal.